15 research outputs found
Investigation of Apatite Mineralization on Antioxidant Polyphosphazenes for Bone Tissue Engineering
Synthetic bone grafts that promote the natural mineralization
process would be excellent candidates for the repair or replacement
of bone defects. In this study, a series of antioxidant-containing
polyphosphazenes were evaluated for their ability to mineralize apatite
during exposure to a solution of simulated body fluid (SBF). All polymers
contained ferulic acid (antioxidant), cosubstituted with different
amino acid esters linked to the polyphosphazene backbone. Differences
in the side groups determined the hydrophobicity or hydrophilicity
of the resulting polymers. All of the polymers mineralized monocalcium
phosphate monohydrate, a type of biological apatite. However, the
mineralization process (the amount of deposition and length of time)
was dependent on the hydrophilicity or hydrophobicity of the polymers.
The polymer–apatite composites were examined by electron scanning
microscopy, X-ray diffraction, Fourier transform infrared spectroscopy,
differential scanning calorimetry, and thermogravametric analysis.
Weight gain data were also obtained. To verify that the nucleation
process was due to the presence of calcium and phosphate, two standard
solutions were prepared: one solution (NaCl solution) contained only
sodium chloride, and the second solution (mSBF) was similar to SBF
except without known crystal growth inhibitors such as Mg<sup>2+</sup> and HCO<sub>3</sub><sup>–</sup>. No mineralization occurred
when the polymers were exposed to the NaCl solution, but mineralization
took place upon exposure to mSBF. The apatite phase produced was hydroxyapatite
(HAp). The mineralization process in mSBF was much more extensive,
with all samples gaining more weight following exposure to SBF. A
similar trend was also found (as in the case of SBF), with the amount
of deposition and length of deposition time depending on the hydrophilicity/hydrophobicity
of the polymer. These results suggest that the nucleation process
is due to calcium and phosphate, and the absence of crystal growth
inhibitors allows for the rapid nucleation of HAp. In both cases,
the mineralization process was favored on hydrophilic surfaces (static
water contact angle of 56–65°) versus hydrophobic surfaces
(71–86°)
Synthesis of Phosphonated Polyphosphazenes via Two Synthetic Routes
Phosphonate and phosphonic acid containing polymers are
of interest
for bone tissue engineering because these species have the ability
to bind hydroxyapatite [Ca<sub>10</sub>(PO<sub>4</sub>)<sub>6</sub>(OH)<sub>2</sub>], which comprises 70 wt % of bone. The synthesis
of phosphoester [−POÂ(OEt)<sub>2</sub>] and phosphonic acid
[−POÂ(OH)<sub>2</sub>] functionalized polyphosphazenes is described.
These polymers could mimic the natural bone healing mechanism, making
them excellent candidates for implantable bone grafts. Two synthetic
protocols have been developed to obtain the polymers, herein referred
to as prior- and post-side-group assembly. Prior assembly required
the synthesis of a phosphonate-containing side group before attachment
to the polyphosphazene backbone through nucleophilic substitution,
whereas post-assembly required the synthesis of a polyphosphazene
containing free amino groups to which the phosphonate can be coupled
by Michael addition after polymer synthesis. The final step for both
routes required the deprotection of the phosphoester to the corresponding
phosphonic acid. The polymers were characterized by <sup>1</sup>H
and <sup>31</sup>P NMR, GPC, and DSC techniques. A six week hydrolysis
study using phosphate buffered saline (PBS) determined their hydrolytic
sensitivity. All the polymers were hydrolytically sensitive, as required
for this purpose, and decomposed ∼2–50% by week six.
The hydrolysis products were analyzed by UV–vis techniques,
and their release was monitored over the course of the experiment.
These results are in agreement with percent solid mass loss data.
In general, all the phosphonic acid polymers hydrolyzed at a faster
rate than their corresponding phosphoester derivatives
Engineered stem cell niche matrices for rotator cuff tendon regenerative engineering.
Rotator cuff (RC) tears represent a large proportion of musculoskeletal injuries attended to at the clinic and thereby make RC repair surgeries one of the most widely performed musculoskeletal procedures. Despite the high incidence rate of RC tears, operative treatments have provided minimal functional gains and suffer from high re-tear rates. The hypocellular nature of tendon tissue poses a limited capacity for regeneration. In recent years, great strides have been made in the area of tendonogenesis and differentiation towards tendon cells due to a greater understanding of the tendon stem cell niche, development of advanced materials, improved scaffold fabrication techniques, and delineation of the phenotype development process. Though in vitro models for tendonogenesis have shown promising results, in vivo models have been less successful. The present work investigates structured matrices mimicking the tendon microenvironment as cell delivery vehicles in a rat RC tear model. RC injuries augmented with a matrix delivering rat mesenchymal stem cells (rMSCs) showed enhanced regeneration over suture repair alone or repair with augmentation, at 6 and 12-weeks post-surgery. The local delivery of rMSCs led to increased mechanical properties and improved tissue morphology. We hypothesize that the mesenchymal stem cells function to modulate the local immune and bioactivity environment through autocrine/paracrine and/or cell homing mechanisms. This study provides evidence for improved tendon healing with biomimetic matrices and delivered MSCs with the potential for translation to larger, clinical animal models. The enhanced regenerative healing response with stem cell delivering biomimetic matrices may represent a new treatment paradigm for massive RC tendon tears
Comparison of the Synthesis and Bioerodible Properties of N-Linked Versus O-Linked Amino Acid Substituted Polyphosphazenes
Insertion morphology of repair, matrix augmentation and matrix/rMSC repair of rat supraspinatus tendons.
<p>Native (<b>CON</b>) tendons (<b><i>A</i></b>) demonstrate a gradual transition from parallel oriented collagenous tendon tissue to bone with a cartilage intermediate. Repair (<b>R</b>) (<b><i>B</i></b>) and matrix augmented (<b>R+S</b>) (<b><i>C</i></b>) insertions have an abrupt transition. Matrix/rMSC repair (<b>R+S+C</b>) (<b><i>D</i></b>) insertions demonstrate a transition and organization similar to the intact tendon. Representative samples at 12-weeks post-surgery stained with Masson’s trichrome. Matrix/sutures are located on the surface plane above the tendon-bone insertion and are present at both 6 (not shown) and 12-weeks. Yellow arrow indicates the bone-tendon axis. Scale bars 200 μm.</p
Imaging and quantification of collagen organization during supraspinatus repair and augmentation.
<p>Slides from native tendon (<b>CON</b>) (<b><i>A</i></b>) and tendons harvested 12-weeks after repair (<b>R</b>) (<b><i>B</i></b>), matrix augmentation (<b>R+S</b>) (<b><i>C</i></b>) and matrix/rMSC (<b>R+S+C</b>) (<b><i>D</i></b>) were stained with picrosirius red and observed under cross-polarized light. Both intact tendon and tendon underlying matrix/rMSC repair demonstrate a high level of birefringence that highlights tissue with highly oriented collagen fiber morphology. Average birefringent signaling from cross-polarized light microscopy of picrosirius red stained slides was converted to an 8-bit grayscale to quantitatively compare the degree of collagen orientation in tendon tissue (<b><i>E</i></b>) from native supraspinatus tendon (<b>CON</b>) and tendons harvested 12-weeks after repair (<b>R</b>), matrix augmented repair (<b>R+S</b>) and cell seeded augmented repair (<b>R+S+C</b>). Both intact tendon and tendon underlying cell seeded augmented repair demonstrate a significantly greater collagen orientation. n = 3 animals per group, * = p<0.05. Scale bars 200 μm, matrix above tendon marked by the yellow grid.</p
Cross-sectional area (A-6-weeks, D-12-weeks), ultimate stress (B-6-weeks, E-12-weeks) and modulus (C-6-weeks, F-12-weeks) of native (CON), repaired (R), augmented supraspinatus (R+S), and matrix/rMSC (R+S+C) tendons at 6 and 12-week post-surgery.
<p>Native tendons possessed significantly less cross-sectional area, greater ultimate stress and greater tensile modulus than all experimental groups at both time points. At 12-weeks the cross-sectional area of matrix/rMSC (R+S+C) tendon was less than the other experimental groups. There was no significant difference in cross sectional area between 6 and 12-week specimens within each experimental group. The ultimate stress (B and E) of matrix/rMSC (R+S+C) tendons was significantly greater than suture repair (R) and augmented repair (R+S) at 6 and 12-weeks. There was no significant difference in ultimate stress between 6 and 12-week specimens within each experimental group with the exception of the repair group (R). The tensile modulus (C and F) of matrix/rMSC tendon was significantly greater than suture repair (R) at 6-weeks and showed a trend of greater modulus than augmented repair (R+S) at 12-weeks. There was no significant difference in tendon modulus between 6 and 12-week specimens within each experimental group with the exception of a trend of increased stiffness for matrix/rMSC. # = p<0.05 (vs. all other groups)</p
Tracing of donor rMSCs during supraspinatus augmentation.
<p>Rats underwent repair with matrices seeded with rMSC stained with PKH26 plasma membrane dye (half-life = 100days). The matrix (<b>White Asterisks</b>) was observed on unstained slides under differential interference contrast microscopy (<b><i>A</i></b>). Only one of three rats at 6-weeks post-surgery demonstrated a faint red fluorescent signal from donor cells (<b><i>B</i></b>). Scale bar 500 μm.</p
Engineered stem cell niche matrices for rotator cuff tendon regenerative engineering - Fig 1
<p>Non-augmented and augmented rat supraspinatus repair model (<b><i>A</i></b>) Modified MasonAllen stitch described by Soslowsky. Purple indicates suture, * indicates areas of stress. (<b><i>B</i></b>) Integrated matrix augmentation model for supraspinatus tendon repair. Green indicates the side of cell seeding in matrix/rMSC group.</p